More than 80% of patients with juvenile myelomonocytic leukemia (JMML) have a mutation in the NF1, RAS, or PTPN11 genes, which encode proteins that are involved in RAS signaling. This information and the observation that children with germline mutations in either NF1 or PTPN11 are predisposed to JMML, strongly implicates hyperactive RAS as playing a central role in the pathogenesis of JMML. In this patient oriented K-22 application, I propose studying these mutated genes in well-characterized cohorts of patients with JMML. First, I will use mutant RAS and PTPN11 as molecular markers of disease activity in patients with JMML. Toward this end, I have developed a novel minimal residual disease assay based on the principles of fluorescence-based amplification of a mutant allele called TaqMAMA. Second, I will study JMML patients who have identified to surprisingly harbor multiple mutations in both PTPN11 and NF1 by analyzing individual subclones that are grown in colony-forming assays. I will determine if multiple mutations are present in each cell or if some cells harbor loss of heterozygosity (LOH) of NF1 while others harbor PTPN11 mutations. Finally, there is considerable cellular heterogeneity in JMML specimens, which complicates detecting aberrant signaling in subpopulations of cells that may be essential for leukemic growth. The hypothesis for aim 3, therefore, is that subsets of cells in the bone marrow of patients with JMML have distinctly perturbed signal transduction networks when exposed to cytokines, such as GM-CSF. I will study existing bone marrow samples obtained from JMML patients at diagnosis, using a novel technique of multi-parameter flow cytometry that permits investigations of signal transduction in subsets of living cells. Using this assay, I will be able to identify discrete populations of cells simultaneously stained with surface markers as well as intracellular phosphoproteins, thereby yielding an assessment of the activation status of Ras effector cascades. The long-term impact of this technology is potentially remarkable, as it might allow investigators to administer a specific inhibitor of a signaling molecule such as Akt, MEK, or mTOR to an individual patient, and perform real-time monitoring of the biochemical target in relevant populations of primary cells. [unreadable] [unreadable] The importance of this research to the general public is that we will develop new tests to follow patients with cancer on therapy, as well as to learn more about the ways that cancer cells communicate -- this may lead to improved medicines with fewer side effects as well as increased survival for patients. [unreadable] [unreadable] [unreadable]